Isolation, (bio)synthetic studies and evaluation of antimicrobial properties of drimenol-type sesquiterpenes of Termitomyces fungi

Macrotermitinae termites have farmed fungi in the genus Termitomyces as a food source for millions of years. However, the biochemical mechanisms orchestrating this mutualistic relationship are largely unknown. To deduce fungal signals and ecological patterns that relate to the stability of this symbiosis, we explored the volatile organic compound (VOC) repertoire of Termitomyces from Macrotermes natalensis colonies. Results show that mushrooms emit a VOC pattern that differs from mycelium grown in fungal gardens and laboratory cultures. The abundance of sesquiterpenoids from mushrooms allowed targeted isolation of five drimane sesquiterpenes from plate cultivations. The total synthesis of one of these, drimenol, and related drimanes assisted in structural and comparative analysis of volatile organic compounds (VOCs) and antimicrobial activity testing. Enzyme candidates putatively involved in terpene biosynthesis were heterologously expressed and while these were not involved in the biosynthesis of the complete drimane skeleton, they catalyzed the formation of two structurally related monocyclic sesquiterpenes named nectrianolins.

A mong the different types of nutritional symbiosis, fungiculture in insects represents one of the most complex symbiotic interactions 1,2 . Only a few insect lineages maintain and manure fungi as nutritional ectosymbionts, similar to human agriculture, with attine ants and Macrotermitine termites being prime examples 1 . In the case of fungus-growing termites, the basidiomycete genus Termitomyces is propagated by termite workers within biomass-containing cork-like structures ("fungus comb") as food fungus, where the fungus colonizes the provided predigested plant material via a dense hyphal network. Subsequently, the fungus forms visible protein, carbohydrate-rich and spore-containing fungal nodules that the termites ingest ( Fig. 1a) 3,4 . While most Termitomyces nodules serve as nutrition for the termites and vector fungal spores (asexual reproduction), other types of nodules occasionally differentiate to become pointy primordia of fruiting bodies (Fig. 1b) 5 . Once pointy primordia mature (Fig. 1c), mushrooms spread sexual spores, which are likely picked up by foraging termites enabling the inoculation of newly founded termite colonies. However, it has been argued that the formation of fruiting bodies likely wastes resources that could otherwise have been allocated to growth of the fungus within the colony. This has led to the hypothesis that termites might actively suppress fruiting body formation of their fungal symbiont by consumption of mushrooms at a primordial stage, and in response the fungus likely evolved gut-resistant asexual spores encapsulated in nodules to ensure its propagation 5 . Thus, it has remained a conundrum in the termite-fungus symbiosis how the reproductive interests of host and symbiont are aligned 1 .
Due to the long co-evolutionary history and the intertwined lifestyles of termites and their food fungus, it has been hypothesized that within the below-ground farming termite system diffusible volatile organic compounds (VOCs) of termites and fungus might play a key role as intra-and interspecific chemical mediators orchestrating the complex symbiosis [6][7][8] .
While studies have showed profound influence of VOCs on behavior, development, and physiology of some social insect species, studies on fungus-growing insects, and particularly termites, have remained rather sparse. Similarly, our understanding of why and when fungi release specific VOCs [9][10][11][12] , and their effects on fungal growth, sexual life cycle 13 , and fungal interactions remains fragmented [14][15][16] . In the fungus-growing termite Odontotermes obesus, a first study indicated that termites are likely able to differentiate between the scent of the food fungus and ascomycetous fungal garden antagonist Pseudoxylaria, and that volatiles emitted by the antagonist, such as the sesquiterpenes aristolene and viridiflorol, are likely triggers of termite hygiene measures 17 . We recently commenced to analyze the VOC profiles emitted by laboratory cultures of Termitomyces cryptogamus 18 retrieved from healthy Macrotermes natalensis colonies 19 . To our surprise, only few terpenoid features were detectable in the volatilome of Termitomyces cultures maintained under laboratory growth conditions, although comparative genomic studies indicated that the fungal mutualist encodes above average numbers of e.g., terpene cyclases responsible for the biosynthesis of e.g., volatile terpenoids 20,21 .
To test the hypothesis that the biosynthesis of terpenoid-based VOCs is life stage dependent and thus tightly regulated in Termitomyces, we analyzed the VOC pattern of Termitomyces in their natural environment and in different growth stages. This unveiled distinct VOC patterns across different biosample types. In mushrooms, sesquiterpenes appeared as dominant features, with drimenol (1) as one of the recurring features emitted from fungal cultures. Targeted purification allowed the isolation of five drimenol derivatives and their characterization by NMR and x-ray crystallography analyses. Total synthesis of a focused drimane library was then pursued and allowed for a comprehensive evaluation of production levels and bioactivity patterns. Subsequent mining of genomic and transcriptomic data uncovered three candidate terpene synthase genes putatively involved in the biosynthesis of drimenol-like terpenes. Heterologous expression of enzyme candidates and bioassays yielded two monocyclic sesquiterpenes, putative biosynthetic shunt products of the drimenol or isolongifolene pathway. Data from our comprehensive study further define how fungal communication might occur by volatiles in the farming symbiosis with termites.
Results and discussion T. cryptogamus mushrooms and fungus comb exhibit different VOC pattern. We collected four different types of samples: (1) fungus-comb interspersed with Termitomyces mycelium, (2) fungus-comb from which a 4-day old Termitomyces mushroom emergesd (3) mushrooms separated from fungus-comb, and (4) axenic fungal agar plate cultures of Termitomyces sp. 153 (closest relative: Termitomyces cryptogamus) from which volatiles were captured on activated charcoal filters using the closed-loop-stripping analysis (CLSA) technique 22,23 . VOCs from mushrooms, fungus comb, and agar plate were collected using a CLSA apparatus over a period of 24 h (Figs. S2-S6, Tables S1-S4). The obtained headspace extracts were analyzed using gas chromatography-mass spectrometry (GC-MS), and data sets were dereplicated and putatively annotated using the National Institute of Standards Mass Spectral Library (NIST 2017). The detected VOCs emitted from comb, mushroom, and axenic cultures were distinct from each other (Fig. 2). Mushroom samples emitted only few aliphatic features with 3-octanone (communication signal, antifungal) 24 , octan-3-ol (phytotoxic and antibacterial), oct-1-en-3-ol 12 (communication signal) and 2-nonenal (antifungal) 25 as the major detectable lipidic constituents. In contrast, a distinct terpenoid signature with sesquiterpenes as the most abundant compound class was detectable within the emitted blend of mushrooms. Overall, β-barbatene, β-cubebene, and brasiladienes were the most dominant features, while drimenol 26 , intermedeol, african-1-ene, and α-amorphene were detected in lower abundances. Here, it was intriguing to note that prior studies on nodules reported the emission of a mostly monoterpene-dominated volatile blend (e.g., α-pinene 27 , camphene, limonene).
In contrast to mushrooms, the volatile blends of fungus comb samples were dominated by aliphatic acids (e.g., hexanoic acid, isovaleric acid, pentanoic acid), ketones (e.g., 3-octanone, 2undecanone), alcohols (e.g., hexanol), and furan derivatives (e.g., 2-pentylfuran, γ-lactones), while β-barbatene was the only detectable terpene feature. The VOC profile changed again when T. cryptogamus isolates were grown on potato dextrose agar (PDA) with drimenol and isolongifolene amongst the most abundant sesquiterpenes, in addition to 2,5-diisopropylpyrazine 28 and 1,2,4trimethoxybenzene, the methylated derivative of the fungal redoxfactor 20 . Intrigued by the emission of the antimicrobial and insect antifeedant sesquiterpene drimenol (1) 29 , we monitored the composition of the volatile blend during fungal growth of Termitomyces sp. T153 on three different media (PDA, cellulose medium, fungus-comb medium) by GC-MS/MS (Figs. S7-S10,  Tables S5-S7). Overall, emission levels of drimenol (1) appeared to reach a maximum after three to four weeks independent of the cultivation medium, and were still detectable even after seven weeks of growth. Interestingly, we were able to assign most of the detectable sesquiterpenes to putative Termitomyces-specific terpene cyclase (TTC) encoding genes (see Table S21) 21 .
T. cryptogamus isolates produce drimanes and drimenols. In addition, secreted metabolites were extracted and analyzed by LC-MS/MS to detect less volatile sesquiterpenoid features during growth on solid media. These studies allowed the identification of more than five molecular ion features with typical sesquiterpenoid MS-fragmentation patterns, which were also detectable by stable isotope feeding experiments using 13 C-enriched acetate (Fig. S11). To clarify their structural assignment, MS-and NMRguided purification of extracts derived from Termitomyces sp. T153 (PDA for 14-21 days) was pursued, which yielded in total five drimenol derivatives (Fig. 3). Chemical structures of isolated compounds were elucidated mainly by comparative 1D and 2D NMR and MS/MS-analysis (Tables S8-S17). The 1 H-NMR spectrum of compound 2 showed three main CH 3 singlets (δ H 0.85, 0.86, and 0.98) and one singlet with slightly higher chemical shift (δ H 1.77) what suggests its attachment to a double bond. This theory was underpinned by finding of only one olefinic methine at δ H 5.53. The main octahydronaphtalene core of the molecule was built up based on the mutual 1 H-1 H COSY correlations between H-1 and H-2, H-2 and H-3, H-5 and H-6, H-6, and H-7. Additional finding of oxy-methine (δ H 3.25) was located to C-3 based on 1 H-1 H COSY correlation with H-2. Position of hydroxymethyl group (δ H 3.73 and 3.85) was solved from 1 H-1 H COSY correlation between H-9 and H-11 and from 1 H-13 C HMBC correlation of H-11 to C-8. Further 1 H-13 C HMBC analysis revealed a correlation of H-3 to C-14 and C-15, H-1 to C-13, and H-7 to C-12 what helped to assign an exact position of CH 3 groups and led to complete structure elucidation. X-ray crystallography of a single crystal of compound 2 (CH 2 Cl 2 /MeOH) secured the structural assignment (Fig. 3). Compounds 3 and 4 showed a similar chemical shift pattern. The main difference was observed in higher chemical shifts of protons at C-1 (δ H 2.74 and 2.28) and C-2 (δ H 2.22 or 2.29 and 1.56) which was caused by a presence of a neighboring keto group at C-3 concluded from appearance of a quaternary carbon with δ C 216.7 for 3 and 215.8 for 4. Presence of a second hydroxymethyl group (δ H 4.38 and 4.43) in 4 was deduced from 1 H-13 C HMBC correlations between H-12 and C-9, H-7 and C-12. Closer NMR analysis of compound 5 revealed similar structure core than 2 with hydrofuranone ring in position C-8 and C-9. For compound 6, a dehydroxylated core in C-3 with δ H 1.22 and 1.41 was deduced. No presence of a double bond in 1 H-NMR was observed. However, a chemical shift of H-7 (δ H 3.76) and δ C 72.9 for C-7 and δ C 76.0 for C-8 suggested that the alkene underwent a dihydroxylation. Structural assignment of isolated compounds 2-6 was further supported by comparison of analytical data with literature reports (compound 2: Marasmius oreades 30 , compound 3: Clitocybe conglobate 31 , compound 4: Phellinidium sulphurascens 32 , compound 5: Peniophora polygonia 33 , compound 6: synthesis of stereoisomers 34,35 and synthetic derivatives (vide infra).
By using the isolated and synthetic compounds as metabolomic references, we then tested if drimenol derivatives were also produced by two other Termitomyces isolates. After two weeks of growth on PDA, we found the characteristic GC-MS and HRMS/ MS signatures of compounds (1-6) in isolates T112 and J132 (Figs. S12, S13), but we were unable to identify any matching signal for derivatives 7-19 in organic extracts of Termitomyces cultures. Thus, we assume that only a subset of drimenols might be characteristic natural products for members of the fungal genus Termitomyces.
Identification and analysis of putative drimenol synthases. Drimane-and drimenol-type sesquiterpenes are a large group of natural products containing a C 15 bicyclic skeleton that have been identified from various plants 45 , animals, the two major division within the fungal kingdom (Basidiomycota and Ascomycota) 26,46,47 , and bacteria 48,49 . The biosynthesis of drimenol (1) has also gained much attention as drimenol can be formed via both class I and class II catalytic mechanisms. While drimenol synthases identified from plants were characterized as class I terpene cyclases (TCs), bacterial drimenol synthases were identified as class II sesquiterpene cyclases [50][51][52] . In contrast, dedicated bifunctional haloacid dehalogenase-like (HAD-like) terpene cyclases were reported from Aspergillus species 53,54 and most recently from Antrodia cinnamomea 55 . Hence, we performed a phylogenomic survey of members of the genus Termitomyces to identify putative candidate sequences, but were unable to relate any of the terpene synthase sequences identified in our previous study to reported sequences involved in the drimenol or isolongifolene biosynthesis (Tables S20, S21) 21 . We then performed a manual BLAST search against predicted protein sequences from different Termitomyces genomes using the sequence of the characterized HAD-like terpene cyclase AstC protein sequence (Gene ID AORIB40_05908) from Aspergillus oryzae as query sequence. In total, we identified three orthologous sequences (DS1-3), which contain motifs of type I and type II terpene synthases. While sequence DS1 is encoded in drimenol-producing Termitomyces strains T153, T112, and J132, sequences DS2 and DS3 were encoded in four (DS2) of a total of six Termitomyces genomes (Figs. S14-S18). We further validated our genome mining results by sequence alignments, which uncovered the characteristic conserved motifs (DDXXE, DxDTT, QW) important to perform ionization-dependent type I and protonation-dependent type II cyclisation reactions, thus indicating bifunctional properties of the enzyme (Figs. S14-S18). Notably DS3-T153 (57.2 kDa) lacked a hydrophobic C-terminal sequence, which was present in the sequence of DS1-T153 (59.5 kDa) and DS2-T153 (66.0 kDa).
We then revisited the putative formation of oxidized sesquiterpenes 2-6 31,56 , and hypothesized that either drimenol (1) or drimenyl pyrophosphate should serve as their biosynthetic precursor (Fig. 5) 57 . For compound 2, an enzymatic hydroxylation of 1 at C-3, appeared most likely, which would yield after a second oxidation step, similar to what was recently shown in Aspergillus calidoustus 54 and A. cinnamomea 55 . At this stage, we also considered that epoxy-FPP 21 could serve as a substrate for the yet unknown DS, which would result-after cyclization and hydrolysis-in the formation of compound 2 as has been shown for the fungal meroterpenoid terretonin 58 . Subsequent enzymatic oxidation of diol 2 at C-12 could then yield drimentriol (II), which after additional oxidation at C-3 affords 4. Similarly, a putative drimentriol II might also serve as precursor for aldehyde III, which could then undergo an oxidative transformation to lactone 5 via hemiketal IV formation 59 . To gain insights into the observed enzymatic oxygenation patterns, we also surveyed the genomic environment up-and downstream (30 kbp) of the putative DS sequences in Termitomyces sp. T153. However, neither orthologous of previously described cytochrome P450 enzymes nor other oxidoreductases were detectable (Tables S22-S24).   (1), drimanol (12) and drimanes (10 and 11) from (+)-sclareolide (7). b Titanocene-catalyzed synthesis of 3-hydroxy-drimanol (18)  Previous RNAseq data analysis of different substrates (fungus comb and Termitomyces mycelium from agar plates) 20,21 clearly indicated that gene sequences of DS1-3-T153 were actively transcribed and coinciding with drimenol production (Fig. S19, Table S25). Thus, the gene sequence of DS2 was obtained after amplification from the respective cDNA (Termitomyces sp. T153) sequence (Tables S25-S27), while codon-optimized transcripts (Termitomyces sp. T153) of DS1 and DS3 were synthesized (BioCat GmbH). Subsequent heterologous production of the histidine-fusion (His6) proteins was performed in E. coli BL21(DE3) using a pET28a vector. However, only DS3-His6  (Fig. 6). However, neither retention time nor fragmentation pattern correlated with data obtained from isolated or synthesized derivatives or the NIST 2017 database. The enzyme reaction was also performed in different buffer systems (Tris-, phosphate-, citrate-buffer) at pH levels of 6 to 8 and in the presence of Mg 2+ or Co 2+ as cofactor, which resulted in all cases in similar product ratios, while only the addition of Mn 2+ as cofactor inhibited the enzyme reaction (Figs. S25-27). In a previous study on Termitomyces terpene cyclase 15 (TTC15-T153) from strain T153, the characterized enzyme was versatile in substrate acceptance and product profile 21 . Thus, we included GPP and GGPP as well as FPP-epoxide and farnesol-epoxide as epoxidized substrates can be cyclized by type II terpene synthases (e.g., oxidosqualene cyclase) resulting in a hydroxy group at the respective position 60 . However, pyrophosphates GPP, GGPP, and FPP-epoxide yielded only dephosphorylated starting material, while farnesol-epoxide remained unreacted (Fig. S27).
To determine the chemical structures of the heterologous products 22 and 23, the enzyme reaction was performed again with 58 mg purified DS3-His6 enzyme (enzyme derived from a 4 L induced E. coli BL21 (DE3) pET28a (+) culture, Fig. S28    Nectrianolins were discovered in 2017 from the phytopathogenic fungus Nectria pseudotrichia 62 , and show structural relation to (R)-trans-γ-monocyclofarnesol that was originally isolated from medicinal mushroom Phellinus linteus 55,63 and include several synthetic compounds 64,65 . As the candidate enzyme DS3-His6 offered neither drimenol nor any related C 15 bicyclic skeleton, we deduced that DS3-His6 likely acts either as a (R)trans-γ-monocyclofarnesol synthase (similar to AncA) or as a dysfunctional drimenol synthase with similar mechanistic activity (Fig. 7). In addition, at this stage it cannot be fully excluded that phosphorylated precursor of 22-23 could serve as precursor for drimenols through a subsequent enzymatic reaction as originally proposed for (+)-trans-γ-monocyclofarnesol 63 . With isolated compounds at hand, we then reanalyzed if Termitomyces produced monocyclofarnesol volatiles in detectable amounts. However, we were unable to identify any matching signal for nectrianolins or (R)-trans-γ-monocyclofarnesol within the prior recorded MS-spectra, suggesting that these compounds are indeed likely to be transient biosynthesis intermediates.
Drimenols show structure-dependent antibacterial activity. Due to the widespread occurrence of drimane-and drimenoltype sesquiterpenes in nature, it is reasonable to assume that they fulfill important, yet mostly unknown, eco-physiological roles to the producer [46][47][48][49] . Furthermore, many derivatives display biologically and pharmacologically interesting activities such as anti-inflammatory, cytotoxic or antimicrobial activity 66,67 , as well as antifeedant activities against insects in case of drimane dialdehydes 68 . Thus, we evaluated the activity of the compounds described in this study using ecology-based assays. However, we observed neither growth modulating effects on the producer Termitomyces sp. T153 (Fig. S29), nor was any antifeedant activity against the model organism Spodoptera littoralis observed 69 . We then evaluated their activity against a panel of fungal and bacterial test strains and were intrigued to note that drimenol 1 and aldehyde derivative 11 were most active, with moderate antifungal activities against Penicillium notatum and Candida albicans (Fig. 8)

Conclusion
Driven by our goal to entwine the volatile-based communication activities within the complex multi-partner symbiosis of the fungus-growing termite M. natalensis, we comparatively analyzed the emitted volatiles blends of mushrooms of different Termitomyces isolates associated with this termite species, along with fungus comb and axenic cultures. We demonstrated that mushrooms produce characteristic VOC patterns, which differ from nodules and axenic lab culture of the fungal mutualist. The combination of targeted isolation and synthesis of ten drimenol derivatives allowed us to verify the detected sesquiterpenoid features and study their antimicrobial effects, with drimenol (1) and isolated derivative (2). RNAseq-assisted analysis led to the identification of three putative drimenol synthase candidates. However, only one active enzyme variant (DS3-His6) was obtained by heterologous enzyme production, and which was found to catalyze the formation of cyclofarnesyl derivatives nectrianolin 22 and 23. Drimane sesquiterpenes exhibit diverse biological activities (e.g., cytotoxic, antifeedant, insecticidal, antimicrobial) and this broad activity spectrum could play a key role in the fungusgrowing termite system by mediating communication between termites and their fungal crop, but also by regulating growth of other microbes than Termitomyces sp. in the fungus comb. This is likely to be context-dependent; for example, drimenol was mainly emitted from mushrooms and lab cultures. This metabolite exhibits antifungal and germination inhibiting activity 29 , which could aid in nest defense under natural conditions. As mushroom formation in nature has not yet been observed for fungal strains actively farmed by M. natalensis colonies, we hypothesize that termite workers may be able to detect morphology-dependent changes in the volatilome of their mutualistic fungus, including drimenols, and might respond by actively suppressing the energyconsuming formation of mushrooms by grooming or feeding activities. However, once Termitomyces mushrooms manage to develop in the absence of termites, the emission of this antimicrobial volatile blend may aid in protection from bacterial infection of the fruiting body. The biochemical findings of this study provided additional evidence for the importance of terpenes in this symbiotic relation and pave the way to unravel the

Material and methods
Collections. Fungus comb material was excavated from mature Macrotermes natalensis colony MN187 (S24 40.434 E28 48.275) and fungus comb carrying pointy nodules were collected from Macrotermes natalensis colony MN188 (S24 40.512 E28 48.260). Fungus combs were carefully transferred to sterile plastic containers supplied with a wet filter paper, where they were kept at 4°C in the dark. Containers were regularly inspected for contaminations with non-Termitomyces fungi 5,20,21 . Termitomyces mushrooms grew from pointy nodules.
Volatile analysis. The emitted metabolites of all samples were collected shortly after collection using the Closed Loop Stripping Apparatus (CLSA) headspace technique (Fig. S2) 70,71 . Samples were placed in a closed chamber under circulating air stream passed through a charcoal filter (Chromtech GmbH, Idstein, Precision Charcoal Filter, 5 mg) for 24 h. The filter was extracted using 50 µl CH 2 Cl 2 and the extracts were analyzed by GC-MS. Identification of compounds was performed by comparing the mass spectra to NIST spectra libraries and retention indices (I) from the literature. Structures were only annotated if both identifiers fit (Figs. S1-S9, Tables S1-S9).
Cultivation. Termitomyces spp. were cultivated on potatodextrose agar (PDA, 25 mL per plate, standard 15 × 90 mm) for a maximum of four weeks at room temperature. Sub-culturing was done by scraping mycelium from half a plate, mixing with 10 mL sterile PBS and spreading 500 µL suspension per plate or used as inoculum of liquid culture broth. Three minimal media containing different carbon sources were inoculated with mycelium and cultivated at room temperature for a given time.
Cultivation on 13 C-isotope enriched medium. Termitomyces sp. T153 was grown on reduced PDA medium (8.7 g/L) supplemented with 20 mM 13 C-enriched sodium acetate (1-13 C or 1,2-13 C, Sigma-Aldrich, USA) for three weeks. Afterward plates were cut into small pieces and extracted with CH 2 Cl 2 overnight. The solvent was filtered and dried under vacuum. Crude extracts were dissolved in MeCN and analyzed using HR-LC-MS ( Figure S10).
Isolation procedures. Termitomyces sp. T153 was cultivated on 40 PDA agar plates (2 L PDA) at room temperature for two and four weeks, respectively. After 2 weeks of incubation, 20 plates were cut in small pieces and soaked in CH 2 Cl 2 overnight. Extracts were filtered and organic solvent evaporated. The crude extracts were dissolved in cyclohexane and fractionated on Chromabond SiO 2 column with a gradient from 100% cyclohexane to 100% EtOAc for 10 min using flash chromatography. A second purification step was performed using reverse phase HPLC with a phenyl-hexyl column and a gradient starting at 40% MeCN isocratic for 5 min followed by a gradient from 40% MeCN to 50% MeCN for 14 min. Final purification of compound 4 was performed using a phenyl-hexyl column and an isocratic gradient with 35% MeCN/65% H 2 O + 0.1% FA for 20 min. Compounds 5 and 6 were purified using a Luna C18 column and 35% MeCN/ 65% H 2 O + 0.1% FA or 45% MeCN/65% H 2 O + 0.1% FA, respectively.
After 4 weeks, the remaining plates were cut in small pieces and soaked in MeOH overnight. After filtration and solvent evaporation, organic extracts were redissolved in 20% MeOH and prepurified by solid phase extraction (SPE; 10 g, Macherey Nagel, Germany) starting from 20% MeOH and eluting by increasing MeOH concentration in 10% steps to 100% MeOH. The 60% MeOH fraction was further purified by reverse phase HPLC on a Luna C18 column starting at 62.5% MeOH for 5 min, applying a gradient from 62.5% MeOH to 100% MeOH for 18 min. Pure compound 3 and compound 2 were obtained after additional using a Luna5u Phenyl-Hexyl column (Phenomenex, 250 × 10 mm) and an isocratic gradient of 45% MeCN over 23 min. X-ray analysis. The intensity data were collected on a Nonius KappaCCD diffractometer, using graphite-monochromated Mo-K α radiation. Data were corrected for Lorentz and polarization effects; absorption was considered on a semiempirical basis using multiple scans [72][73][74] . The structure was solved by direct methods (SHELXS) and refined by full-matrix least-squares techniques against Fo 2 (SHELXL-2018). The hydrogen atoms bonded to the hydroxyl groups O1, and O2 of the two independent molecules A and B of 2 were located by difference Fourier synthesis and refined isotropically. All other hydrogen atoms were included at calculated positions with fixed thermal parameters. All nonhydrogen atoms were refined anisotropically 75  Genome mining. To improve the genome assembly and annotation of Termitomyces sp. T153 (GCA_018296165.1) the strain T153 was sequenced using Oxford Nanopore Technology (Oxford Nanopore Technologies, Oxford, UK). For this, DNA was extracted from Termitomyces sp. T153 cultures grown in PDB for 1 week (28°C, 300 rpm). The mycelium was filtered, frozen at -80°C and lyophylized for 24 h. The freeze-dried material was ground to a fine powder and 10 mg was used for subsequent DNA extraction using the DNA plant kit (Qiagen). For better purity, DNA was precipitated with ice cold iPrOH, centrifuged and the pellet was washed with 70% EtOH and dried by compressed air. For analysis DNA was dissolved in water. The MinION sequencing library was prepared using the Rapid DNA sequencing kit (SQK-RAD4) according to the manufacturer. DNA sequencing was performed on a MinION Mk1B sequencing device equipped with a R9.4.1 flow cell, which was prepared and run according to the manufacturer. Nanopore sequencing raw data was generated using MinKNOW software version 4.0.20 (Oxford Nanopore Technologies) and was base-called and trimmed using Guppy version 4.2.2 (Oxford Nanopore Technologies). The resulting fastq files were filtered using Nanofilt. A hybrid de novo genome assembly, combining BGISeq and Oxford Nanopore data, was performed using MaSuRCA version 3.4.1. The resulting draft assembly was then polished with the accurate Illumina reads using the POLCA genome polisher. Putative drimenol synthases in predicted proteins of Termitomyces sp. T153 were identified from BLAST search against AstC protein (Gene ID AORIB40_05908). The genomic environment of putative drimenol synthase genes (DS1-3) in Termitomyces sp. T153 was analyzed for the presence of putative natural product biosynthetic enzyme genes 30.000 bp up-and downstream of the DS1-3 sequences (Tables S20-25).
Generation of DS2 sequence. We reanalyzed the relative expression levels of TC-related gene sequences in RNAseq data obtained from axenic Termitomyces sp. 153 and J132, as well as fresh and old fungus comb and nodules on which young workers feed 21 . The transcript sequence of DS2 was obtained by PCR from a cDNA template. RNA of Termitomyces sp. T153 was extracted from frozen mycelium of a culture grown on PDA plate cultures for~2 weeks using the "Isolate II RNA plant" Kit (Bioline). cDNA was obtained using the following protocol: 2 µg RNA, 2 µL Oligo d(T) 23 VN primer (NEB), 1 µL dNTP mix (25 mM each, biotech rabbit) and adjustment with water to a volume of 18 µL; incubation for 5 min at 65°C, followed by addition of 6 µL RT buffer, 1 µL RT enzyme (Thermo, Maxima H Minus), 1 µL RNase-Out (Invitrogen) and 4 µL water; cDNA synthesis was performed for 2-3 h at 46°C finalized by an inactivation step at 85°C for 5 min.
Cloning protocol. Native cDNA of DS2 was used as template for PCR amplification. The restriction site NheI/BamHI was added to forward and the restriction site HindIII added to reverse primer sequence for ligation into pET28a (+) vector (Table S25) Gibson Assembly (NEBuilder Hifi DNA assembly, NEB) was performed with vector-insert ratio 1:2 according to protocol and incubated for 1 h at 50°C. The mixture was transformed in DH5α/BL21 cells and positive transformants were used for heterologous expression studies with ampicillin (100 µg/mL) as selecting antibiotic.
Heterologous production of DS1-3. An overnight culture of E. coli BL21 (DE3) containing the prepared plasmids was grown in LB medium (kanamycin 60 µg/mL). For expression experiments, a 1 mL o/n culture was used to inoculate 50 mL LB medium (0.4% glycerol, 60 µg/mL kanamycin). The culture was incubated first at 37°C (180 rpm shaking) until a density of OD 600 0.4, then incubated at 16°C (160 rpm) for 1 h, and after that induced with 0.1 mM IPTG followed by growth over night (16°C). Cells were centrifuged at 4°C for 15 min (16.000 rpm) and resuspended in lysis buffer (2 mL, 200 mM NaCl, 100 mM Tris-HCl, 5% glycerol, pH 7.0). Homogenized cells were sonicated 3 × 2 min at 0°C and again centrifuged as mentioned above. The supernatant was defined as soluble fraction. The pellet was resuspended in 1 mL urea lysis buffer (500 mM NaCl, 100 mM Tris-HCl, 7 M urea, pH 8) and incubated at room temperature for 5 min. After another centrifugation step, supernatant was transferred to a new tube and defined as pellet fraction. Samples were adjusted to a concentration of 5 mg/mL, diluted with 4× Laemmli sample buffer (Biorad), and incubated at 90°C for 10 min. For SDS-PAGE, precast gels "Any kD Mini-PROTEAN TGX" (Biorad) were used.
Western blot analysis. an SDS-PAGE was transferred to a nitrocellulose membrane using the iBlot2 dry blotting system (ThermoFisher Scientific). Afterward, the membrane was shaken in blocking solution (TBS-T + 5% milk powder) for 1 h. Then, 40 µL of His-Probe antibody (HIS.H8, Santa Cruz Biotechnology) was added and incubated overnight under mild shaking. The resulting membrane was washed 3x with TBS-T, added to blocking solution and 10 µl of the secondary antibody (m-IgGκ BP-HRP, Santa Cruz Biotechnology). After 2 h shaking incubation, the membrane was washed and the chemiluminescence signal of HRP was visualized by adding WesternSure chemiluminescent substrate (LI-Cor Bioscience, according to protocol) and visualization with FUSION FX imaging system (Vilber Lourmat GmbH).
GC-MS analysis of enzyme assay products. Analysis of DS assay products was conducted using an Agilent 6890 Series gas chromatograph coupled to an Agilent 5973 quadrupole mass selective detector (interface temp, 270°C; quadrupole temp, 150°C; source temp, 230°C; electron energy, 70 eV). Terpenes were separated using a ZB5 column (Phenomenex, Aschaffenburg, Germany, 30 m × 0.25 mm × 0.25 µm) and He as carrier gas (flow, 2 mL/ min). The sample (1 µL) was injected without split at an initial oven temperature of 45°C. The temperature was held for 2 min and then increased to 280°C with a gradient of 7°C/min, and then further increased to 330°C with a gradient of 60°C/min and a hold of 1 min.
Enzyme assays. Soluble protein fractions of DS1-MPB, DS2-MPB, His6-DS3, and a control culture carrying an empty pET28a(+) vector were prepared from 100 mL of E. coli and enzyme reactions were set up in glass vials. Terpene assays were performed using single purified enzymes (DS1-/DS2-MBP and His6-DS3) and combinations thereof. Enzyme assay using versions of DS1-DS3 was set up by mixing 50 µL soluble protein extract, 5 µL 10 mM FPP, 5 µL 200 mM MgCl 2 , 40 µL assay buffer (10 mM Tris-HCl, 2 mM DTT, 10% glycerol, pH 7.2). Reaction mixture was covered with a layer of 100 µL hexane and incubated at 30°C for 2 h. Samples were afterwards vortexed for 2 min, frozen in liquid nitrogen and hexane phase was taken directly for GC-MS analysis.
Upscaling of enzyme assay. Overnight culture of E. coli BL21 (DE3) pET28-DS3 (4 × 20 mL) were grown in LB (kanamycin 60 µg/mL) and used to inoculate 4 × 1 L LB medium (0.4% glycerol, 60 µg/mL kanamycin). The cultures were first incubated at 37°C and 180 rpm shaking until the cells grew to a density of OD 600 0.4, then kept at 16°C (160 rpm) and induced with 0.1 mM IPTG after 1 h. After cultivation o/n cells were centrifuged (4°C, 15 min, 16.000 rpm) and resuspended in IMAC FPLC lysis buffer (15 mL per 1 L culture, 500 mM NaCl, 50 mM Tris-HCl, 50 mM imidazol pH 7.4). Homogenized cells were sonicated 3× for 2 min (0°C) and centrifuged (4000 rcf, 30 min). The supernatant was purified by fast protein liquid chromatography (FPLC) on a NGC Quest 10 Plus Chromatography System from Bio-Rad with Nuvia IMAC Ni-Charged 5 mL column (BioRad) equilibrated with IMAC FPLC buffer. A gradient of 0% to 50% buffer B (50 mM Tris pH 7.4, 500 mM NaCl, 500 mM imidazole) was applied over 10 column volumes. Putative target fractions were checked by SDS-Page for correct size and potential impurities. Fractions containing protein DS3 were combined and concentrated with MWCO 5000 at 4000 rcf, 4°C ca 3 h to a volume of 2 mL and diluted with standard protein buffer to 60 mL. Purified protein solution (60 mL) was diluted with assay buffer (48 mL) and incubated in the presence of FPP (115 mg synthesized FPP dissolved in 6 mL 50 mM NH 4 HCO 3 buffer), and 200 mM MgCl 2 solution (6 mL), were incubated at 30°C overnight. The reaction was quenched by addition of cyclohexane (120 mL), and then filtered through a short pad of Celite. The organic layer was separated and the remaining aqueous layer was extracted 3× with cyclohexane. Combined organic layers were washed with brine, dried over MgSO 4 , filtered, and concentrated in vacuo to give 90.4 mg of the crude extract. Compounds were purified by flash chromatography (cyclohexane/EtOAc) and preparative HPLC purification (MeCN/ ddH 2 O + 0.1% FA) and yielded compound 22 Antimicrobial assay. Disc diffusion assays of standard microbial strains were performed following the Clinical and Laboratory Standards Institute guidelines. Antimicrobial activities (zones of inhibitions) were monitored in mm and compared to positive and negative controls. For Termitomyces agar diffusion assays, PDA plates were inoculated with strain T153. After 1 day, paper discs soaked with 10 µl compound solution (1 mg/mL in MeOH) were placed in the middle of inoculated Termitomyces plates and incubated at room temperature. Plates and zones of inhibition were monitored daily for 12 days.